The field of the present invention is plant molecular biology, especially as related to genetically modified plants with resistance to herbicide. Specifically, the present invention relates to transgenic plants in which herbicide resistance is achieved by introducing a coding sequence which determines an herbicide resistance protoporphyrinogen oxidase (PPO) which is expressed in chloroplasts and mitochondria. Such transgenic crop plants are useful in fields where it is desired to spray herbicide to improve crop yield.
A major concern with the use of herbicides for weed control is the selection of resistant populations. To date, over 300 different herbicide-resistant weed biotypes have been identified worldwide (see weedscience.com on the internet). Numerous factors influence the likelihood of herbicide-resistance evolution in a weed population, and certain herbicides are more prone to resistance evolution than are others. For example, populations of 95 weed species have been reported with resistance to herbicides that inhibit acetolactate synthase (ALS), whereas evolved resistance to herbicides that inhibit protoporphyrinogen oxidase (PPO) has been reported for only three weeds (weedscience.com website), even though these herbicides were first commercialized in the 1960s (1). The first weed to evolve resistance to PPO inhibitors was Amaranthus tuberculatus (waterhemp), an increasingly problematic weed of agronomic production systems throughout the Midwestern United States.
The biosynthetic pathways which lead to the production of chlorophyll and heme share a number of common steps. Chlorophyll is a light harvesting pigment present in all green photosynthetic organisms. Heme is a cofactor of hemoglobin, cytochromes, P450 mixed-function oxygenases, peroxidases, and catalases (see, e.g. Lehninger, 1975, Biochemistry. Worth Publishers, New York), and is therefore a necessary component for all aerobic organisms. The last common step in chlorophyll and heme biosynthesis is the oxidation of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase (referred to herein as PPO or protox) is the enzyme which catalyzes this last oxidation step (Matringe et al. 1989. Biochem. J. 260: 231).
An approach that has been used to isolate biosynthetic genes in metabolic pathways from organisms including the higher eukaryotes is the complementation of microbial (auxotrophic) mutants deficient in the activity of interest. For this approach, a library of cDNAs from the higher eukaryote is cloned in a vector that can direct expression of the cDNA in the microbial host. The vector is then transformed or otherwise introduced into the mutant, and colonies are selected that no longer require the nutritional supplementation of interest. Microbial mutants believed defective in PPO activity have been described (e.g. E. coli (Sasarman et al. 1979. J. Gen. Microbiol. 113: 297), Salmonella typhimurium (Xu et al. 1992. J. Bacteriol. 174: 3953), and Saccharomyces cerevisiae (Camadro et al. 1982. Biochem. Biophys. Res. Comm. 106: 724.
The use of herbicides to control undesirable vegetation such as weeds or plants in crops has become common, with the relevant market exceeding a billion dollars a year. Despite extensive herbicide use, weed control remains a significant and costly problem for farmers. Since various weed species are resistant to herbicides, the production of effective herbicides becomes increasingly important, as is the development of agronomically important plants which are resistant to one or more herbicides.
The PPO enzyme is the target of a variety of herbicides. PPO-inhibiting herbicides include many different structural classes of molecules (Duke et al. 1991. Weed Sci. 39: 465; Nandihalli et al. 1992. Pesticide Biochem. Physiol. 43: 193; Matringe et al. 1989. FEBS Lett. 245: 35; Yanase and Andoh. 1989. Pesticide Biochem. Physiol. 35: 70). These herbicidal compounds include the diphenylethers {e.g. lactofen, (±)-2-ethoxy-1-methyl-2-oxoethyl 5-{2-chloro-4-(trifluoromethyl)phenoxy}-2-nitrobenzoate; acifluorfen, 5-{2-chloro-4-(trifluoromethyl)phenoxy}-2-nitrobezoic acid; its methyl ester; or oxyfluorfen, 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluorobenzene)}, oxidiazoles, (e.g. oxidiazon, 3-{2,4-dichloro-5-(1-methylethoxy)phenyl}-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one), cyclic imides (e.g. S-23142, N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide; chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide), phenyl pyrazoles (e.g. TNPP-ethyl, ethyl 2-{1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy}propionate; M&B 39279), pyridine derivatives (e.g. LS 82-556), and phenopylate and its O-phenylpyrrolidino- and piperidinocarbamate analogs. Many of these compounds competitively inhibit the normal reaction catalyzed by the enzyme, apparently acting as substrate analogs.
Additional herbicides of interest include 3-Phenyluracils of formula I
wherein R1 is methyl or NH2; R2 is C1-C2-haloalkyl; R3 is hydrogen or halogen; R4 is halogen or cyano; R5 is hydrogen, cyano, C1-C6-alkyl, C1-C6-alkoxy, C1-C4-alkoxy-C1-C4-alkyl, C3-C7-cycloalkyl, C3-C6-alkenyl, C3-C6-alkynyl or benzyl which is unsubstituted or substituted by halogen or alkyl; and R6, R7 independently of one another are hydrogen, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-alkenyl, C3-C6-alkynyl, C3-C7-cycloalkyl, C3-C7-cycloalkenyl, phenyl or benzyl, where each of the 8 abovementioned substituents is unsubstituted or may be substituted by 1 to 6 halogen atoms and/or by one, two or three groups selected from: OH, NH2, CN, CONH2, C1-C4-alkoxy, C1-C4-haloalkoxy, C1-C4-alkylthio, C1-C4-haloalkylthio, C1-C4-alkylsulfonyl, C1-C4-haloalkylsulfonyl, C1-C4-alkylamino, di(C1-C4-alkyl)amino, formyl, C1-C4-alkylcarbonyl, C1-C4-alkoxycarbonyl, C1-C4-alkylaminocarbonyl, di(C1-C4-alkyl)aminocarbonyl, C3-C7-cycloalkyl, phenyl and benzyl; or R6, R7 together with the nitrogen atom form a 3-, 4-, 5-, 6- or 7-membered saturated or unsaturated nitrogen heterocycle which may be substituted by 1 to 6 methyl groups and which may contain 1 or 2 further heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur as ring members, and their agriculturally acceptable salts (as described in the patent application PCT/EP 01/04850.
Application of PPO-inhibiting herbicides results in the accumulation of protoporphyrinogen IX in the chloroplast and mitochondria, which is believed to leak into the cytosol where it is oxidized by a peroxidase. When exposed to light, protoporphyrin IX causes formation of singlet oxygen in the cytosol and the formation of other reactive oxygen species, which can cause lipid peroxidation and membrane disruption leading to rapid cell death (Lee et al. 1993. Plant Physiol. 102: 881).
Not all PPO enzymes are sensitive to herbicides which inhibit plant PPO enzymes. Both the Escherichia coli and Bacillus subtilis PPO enzymes (Sasarmen et al. 1993. Can. J. Microbiol. 39: 1155; Dailey et al. 1994. J. Biol. Chem. 269: 813) are resistant to these herbicidal inhibitors. Mutants of the unicellular alga Chlamydomonas reinhardtii resistant to the phenylimide herbicide S-23142 have been reported (Kataoka et al. 1990. J. Pesticide Sci. 15: 449; Shibata et al. 1992. In Research in Photosynthesis, Vol. III, N. Murata, ed. Kluwer: Netherlands. pp. 567-70). At least one of these mutants appears to have an altered PPO activity that is resistant not only to the herbicidal inhibitor on which the mutant was selected, but also to other classes of protox inhibitors (Oshio et al. 1993. Z. Naturforsch. 48c: 339; Sato et al. 1994. In ACS Symposium on Porphyric Pesticides, S. Duke, ed. ACS Press: Washington, D.C.). A mutant tobacco cell line has also been reported that is resistant to the inhibitor S-21432 (Che et al. 1993. Z. Naturforsch. 48c: 350). Auxotrophic E. coli mutants have been used to confirm the herbicide resistance of cloned plant PPOs.
There is a need in the art for effective and efficient herbicide resistance genes in plants, especially crop plants, so that application of herbicide to cultivated fields results in good growth of the desired crop plants and eradication (or significant reduction) in pest plants, as well as for selectable markers for transgenic plants, plant cells and plant tissue.